RESUMO
Proteorhodopsins are light-dependent proton pumps that are predicted to have an important role in the ecology of the oceans by supplying energy for microbial metabolism. Proteorhodopsin genes were first discovered through the cloning and sequencing of large genomic DNA fragments from seawater. They were later shown to be widely distributed, phylogenetically diverse, and active in the oceans. Proteorhodopsin genes have not been found in cultured bacteria, and on the basis of environmental sequence data, it has not yet been possible to reconstruct the genomes of uncultured bacterial strains that have proteorhodopsin genes. Although the metabolic effect of proteorhodopsins is uncertain, they are thought to function in cells for which the primary mode of metabolism is the heterotrophic assimilation of dissolved organic carbon. Here we report that SAR11 strain HTCC1062 ('Pelagibacter ubique'), the first cultivated member of the extraordinarily abundant SAR11 clade, expresses a proteorhodopsin gene when cultured in autoclaved seawater and in its natural environment, the ocean. The Pelagibacter proteorhodopsin functions as a light-dependent proton pump. The gene is expressed by cells grown in either diurnal light or in darkness, and there is no difference between the growth rates or cell yields of cultures grown in light or darkness.
Assuntos
Alphaproteobacteria/química , Rodopsina/química , Rodopsina/metabolismo , Alphaproteobacteria/genética , Alphaproteobacteria/metabolismo , Biologia Marinha , Filogenia , Plâncton/química , Plâncton/genética , Plâncton/metabolismo , Rodopsina/genética , Rodopsinas Microbianas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The SAR11 clade consists of very small, heterotrophic marine alpha-proteobacteria that are found throughout the oceans, where they account for about 25% of all microbial cells. Pelagibacter ubique, the first cultured member of this clade, has the smallest genome and encodes the smallest number of predicted open reading frames known for a free-living microorganism. In contrast to parasitic bacteria and archaea with small genomes, P. ubique has complete biosynthetic pathways for all 20 amino acids and all but a few cofactors. P. ubique has no pseudogenes, introns, transposons, extrachromosomal elements, or inteins; few paralogs; and the shortest intergenic spacers yet observed for any cell.
Assuntos
Alphaproteobacteria/genética , Genoma Bacteriano , Água do Mar/microbiologia , Alphaproteobacteria/classificação , Alphaproteobacteria/isolamento & purificação , Alphaproteobacteria/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Composição de Bases , Evolução Biológica , Carbono/metabolismo , Biologia Computacional , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Intergênico , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Oceanos e Mares , Fosfatos/metabolismo , Filogenia , Seleção Genética , Fator sigma/genética , Timidilato Sintase/genéticaRESUMO
Gene site saturation mutagenesis (GSSM) technology is applied for the directed evolution of a nitrilase. The nitrilase effectively catalyzes the desymmetrization of the prochiral substrate 3-hydroxyglutaronitrile to afford (R)-4-cyano-3-hydroxybutyric acid, a precursor to the valuable cholesterol-lowering drug Lipitor. The discovered wild-type enzyme effectively performs the reaction at the industrially relevant 3 M substrate concentration but affords a product enantiomeric excess of only 87.6% ee. Through GSSM, a mutagenesis technique that effects the combinatorial saturation of each amino acid in the protein to each of the other 19 amino acids, combined with a novel high-throughput mass spectroscopy assay, a number of improved variants were identified, the best of which is the Ala190His mutant that yields product enantiomeric excess of 98.5% at 3 M substrate loading and a volumetric productivity of 619 g L-1 d-1.
Assuntos
Aminoidrolases/química , Aminoidrolases/genética , Substituição de Aminoácidos , Aminoidrolases/metabolismo , Hidroxibutiratos/síntese química , Mutagênese Sítio-Dirigida , EstereoisomerismoRESUMO
The hyperthermophile Nanoarchaeum equitans is an obligate symbiont growing in coculture with the crenarchaeon Ignicoccus. Ribosomal protein and rRNA-based phylogenies place its branching point early in the archaeal lineage, representing the new archaeal kingdom Nanoarchaeota. The N. equitans genome (490,885 base pairs) encodes the machinery for information processing and repair, but lacks genes for lipid, cofactor, amino acid, or nucleotide biosyntheses. It is the smallest microbial genome sequenced to date, and also one of the most compact, with 95% of the DNA predicted to encode proteins or stable RNAs. Its limited biosynthetic and catabolic capacity indicates that N. equitans' symbiotic relationship to Ignicoccus is parasitic, making it the only known archaeal parasite. Unlike the small genomes of bacterial parasites that are undergoing reductive evolution, N. equitans has few pseudogenes or extensive regions of noncoding DNA. This organism represents a basal archaeal lineage and has a highly reduced genome.